Reporter genes have become an invaluable tool in studies of gene expression. They are widely used in biomedical and pharmaceutical research and also in molecular biology and biochemistry. A key feature in reporter assays is an expression cassette comprising a coding region for marker of interest and a transcriptional regulatory region, i.e., a promoter regulatory region, that drives expression of the reporter. Any agent can then be tested for direct or indirect effects on the promoter, thereby identifying agents with potential value in directing control of products naturally driven by the promoter regulatory region. Common reporters are β-galactosidase, β-glucuronidase and luciferase, which rely upon various readouts including luminescence, absorbance and fluorescence.
Luciferase is a generic term for the class of oxidative enzymes used in bioluminescence and is distinct from a photoprotein. The name is derived from Lucifer, the root of which means “light-bearer.” The advantages of a luciferase assay are the high sensitivity, the absence of luciferase activity inside most of the cell types, the wide dynamic range, rapidity and low costs. One example is the firefly luciferase from the firefly Photinus pyralis, and this protein requires no post-translational modification for enzyme activity; it is not even toxic in high concentration (in vivo) and can be used in pro- and eukaryotic cells. Firefly luciferase catalyzes the bioluminescent oxidation of the luciferin in the presence of ATP, magnesium and oxygen.
In many assay formats, it is useful to examine multiple effects using distinct reporter cassettes. This is termed “multiplexing,” i.e., using multiple readouts to simultaneous assess various effects. By using multiplexed assays, one can acquire information much more quickly. While there exist different luciferase enzymes, current assays only permit looking at two different reporters within the confines of a single assay. Thus, there remains a need to develop reagents that permit higher levels of multiplex luciferase assays.